How to calculate the size of my mutant cDNA library? - Science and Technology

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mellowparty
legitimate researcher

Registered: 05/17/09
Posts: 18,467
Last seen: 10 years, 5 months

How to calculate the size of my mutant cDNA library?

#16952179 - 10/02/12 02:20 PM (11 years, 6 months ago)

OK molecular biology problem here. I want to carry out saturation mutagenesis of a gene that codes for a protein with a length of (N) amino acids.

I want my library to contain every plasmid encoding a protein that has 19 amino acid substitutions at every position on the protein (from 0 to N)

How many variant plasmids are there?

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Metathetical
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Registered: 09/08/12
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Re: How to calculate the size of my mutant cDNA library?

[Re: mellowparty]
#16998640 - 10/09/12 02:39 PM (11 years, 6 months ago)

I feel like you would need 19^N plasmids! daunting!
That is assuming you would want to obtain peptides that are all repeats, IE all 19 AA's being the same. Excluding these would bring down your total plasmids. I see it as 19 choices * 19 choices * 19 choices * ... *N
something else to think about is that when N>19 you will always have 1 or more repeats.

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mellowparty
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Registered: 05/17/09
Posts: 18,467
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Re: How to calculate the size of my mutant cDNA library?

[Re: Metathetical]
#16999254 - 10/09/12 04:15 PM (11 years, 6 months ago)

Thats the number of the possible mutant proteins.

Each amino acid is coded for by 3 nucleotides and there are 4³ combinations of nucleotides per codon. 61 cause 3 would be stop codons and there are 20 amino acids so the code is quite redundant. Some times a single nucleotide change is enough to change the amino acid sometimes you need to change all 3.

Is the answer 61^N?

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mellowparty
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Re: How to calculate the size of my mutant cDNA library?

[Re: mellowparty]
#16999268 - 10/09/12 04:17 PM (11 years, 6 months ago)

Btw the mutagenic system makes a mistake every 10⁴ nucleotides.

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Seahawk1
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Registered: 04/12/12
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Re: How to calculate the size of my mutant cDNA library?

[Re: mellowparty]
#17031778 - 10/14/12 08:12 PM (11 years, 6 months ago)

Just use elegans and mutate with EMS. You'll get what you're looking for. Bonus is the gene probably already has mutants you can work with. Also pretty easy to tag the genes/promoters with g/r/yfp to screen.
I might have misunderstood the question; but you'd have a huge library!

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mellowparty
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Re: How to calculate the size of my mutant cDNA library?

[Re: Seahawk1]

1
#17033227 - 10/15/12 12:48 AM (11 years, 6 months ago)

Funny that you mention elegans because it is the only eukaryote that doesn't have this gene haha (its found everywhere else tho from primitive fungi to man). Anyway the idea is to transfect my mutant library in human cancer cells and give them some drugs to see which mutants are resistant. Basically I'm either going to do error-prone PCR or propagate the vector with my gene in a DNA-damage repair-deficient E. coli.

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Seahawk1
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Registered: 04/12/12
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Re: How to calculate the size of my mutant cDNA library?

[Re: mellowparty]
#17045042 - 10/16/12 09:22 PM (11 years, 5 months ago)

Wow, that's crazy they don't have it! I worked with elegans for about two years, I thought the had analogues to everything, hehe! What gene if you don't mind me asking?
Sounds like you need some undergrads!

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mellowparty
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Registered: 05/17/09
Posts: 18,467
Last seen: 10 years, 5 months

Re: How to calculate the size of my mutant cDNA library?

[Re: Seahawk1]
#17046344 - 10/17/12 01:34 AM (11 years, 5 months ago)

TTp

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Edited by mellowparty (10/19/12 11:06 AM)

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